Enzymatic processes for the resolution of enantiomeric mixtures of lactams useful as intermediates in the preparation of taxanes

ABSTRACT

Methods for the enzymatic resolution of mixtures of enantiomers, such as β-lactam compounds, which may be employed as intermediates in the preparation of taxanes bearing a C-13 sidechain containing a heterocyclic or cycloalkyl group, the latter useful in the pharmaceutical field.

This is a continuation of application Ser. No. 08/092,170 filed on Jul.14, 1993 now abandoned.

FIELD OF THE INVENTION

The present invention relates to enzymatic processes for the resolutionof enantiomeric mixtures of compounds useful as intermediates in thepreparation of taxanes, particularly for the preparation of taxanesbearing a C-13 sidechain containing a heterocyclic or cycloalkyl group.

BACKGROUND OF THE INVENTION

Taxanes are diterpene compounds which find utility in the pharmaceuticalfield. For example, taxol analogues containing heterocyclic orcycloalkyl groups on the C-13 sidechain find utility as anticanceragents. Such taxol analogues may be prepared through semi-syntheticroutes, particularly by the coupling of β-lactam or open chainintermediates to the taxane core to form a sidechain at C-13. As thestereochemistry of these analogues may affect their pharmaceuticalactivity, methods allowing efficient stereospecific preparation of theintermediate β-lactam and open chain compounds, as well as the finaltaxane products, are sought in the art.

SUMMARY OF THE INVENTION

The present invention provides efficient methods for the resolution ofenantiomeric mixtures, preferably racemic mixtures, of compounds usefulas intermediates in the preparation of taxanes bearing a C-13 sidechaincontaining a heterocyclic or cycloalkyl group, and thus for thestereospecific preparation of these compounds.

Specifically, the present invention provides a method for the resolutionof a mixture I comprising the enantiomers Ia and Ib, where R¹ is in thecis position relative to R² in both Ia and Ib, or where R¹ is in thetrans position relative to R² in both Ia and Ib: ##STR1## where R¹ ishydroxyl; halo; or --O--C(O)--R⁴, where

R⁴ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl orheterocyclo;

R² is heterocyclo or cycloalkyl; and

R³ is hydrogen; R⁴ ; --C(O)--OR⁴ ; or --C(O)--R⁴,

where R⁴ is independently selected from those groups recited for R⁴above;

comprising the step of contacting said mixture I with an enzyme ormicroorganism capable of catalyzing the stereoselective conversion ofone of said compounds Ia or Ib to a non-enantiomeric form, and effectingsaid conversion.

The present invention also provides a process for the resolution of amixture IV comprising the enantiomers IVa and IVb:

    R.sup.2 --T.sup.a --C(O)--OR.sup.6                         (IVa)

and

    R.sup.2 --T.sup.b --C(O)--OR.sup.6                         (IVb)

where

T^(a) is ##STR2## and T^(b) is ##STR3## or T^(a) is ##STR4## and T^(b)is ##STR5## where R¹ is in the erythro position relative to the group Win both IVa and IVb, or where R¹ is in the threo position relative tothe group W in both IVa and IVb;

W is --NHR³ or --N₃ ;

R¹ is hydroxyl; halo; or --O--C(O)--R⁴, where

R⁴ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl orheterocyclo;

R² is heterocyclo or cycloalkyl;

R³ is hydrogen; R⁴ ; --C(O)--OR⁴ ; or --C(O)--R⁴,

where R⁴ is independently selected from those groups recited for R⁴above; and

R⁶ is hydrogen; or R⁴, where R⁴ is independently selected from thosegroups recited for R⁴ above;

comprising the step of contacting said mixture IV with an enzyme ormicroorganism capable of catalyzing the stereoselective conversion ofone of said compounds IVa or IVb to a non-enantiomeric form, andeffecting said conversion.

Exemplary embodiments for the aforementioned stereoselective conversionsinclude stereoselective hydrolysis, stereoselective esterification,stereoselective transesterification and stereoselective dehalogenation,particularly stereoselective hydrolysis or esterification.

Groups, such as hydroxyl groups, on the compounds of formulae I or IVmay optionally be protected for use in the resolution methods of thepresent invention; such groups may optionally be subsequentlydeprotected.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention are described further as follows.

Cis Enantiomers

The following pair of cis enantiomers may be separated by the enzymaticmethods of the instant invention: ##STR6## that is, enantiomers Ia andIb where R¹ is in the cis position relative to R² in both Ia and Ib.

It is preferred to resolve a mixture of cis enantiomers as describedabove according to the methods of the instant invention.

Trans Enantiomers

The following pair of trans enantiomers may be separated by theenzymatic methods of the instant invention: ##STR7## that is,enantiomers Ia and Ib where R¹ is in the trans position relative to R²in both Ia and Ib.

Erythro Enantiomers

The following pairs of erythro enantiomers may be separated by theenzymatic methods of the instant invention: ##STR8## that is,enantiomers IVa and IVb where R¹ is in the erythro position relative tothe group W in both IVa and IVb.

Threo Enantiomers

The following pairs of threo enantiomers may be separated by theenzymatic methods of the instant invention: ##STR9## that is,enantiomers IVa and IVb where R¹ is in the threo position relative tothe group W in both IVa and IVb.

Preferred Methods for the Resolution of Mixture I

Mixture I, comprising an enantiomeric mixture of β-lactams Ia and Ib, ispreferably resolved by stereoselective hydrolysis, esterification ordehalogenation. A particularly preferred method for the resolution of amixture I comprising the enantiomers Ia(1) and Ib(1): ##STR10## to forma mixture II comprising the compounds IIa(1) and IIb(1): ##STR11## whereR² is heterocyclo or cycloalkyl; and

R³ is hydrogen; R⁴ ; --C(O)--OR⁴ ; or --C(O)--R⁴,

where R⁴ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl orheterocyclo;

comprises one of the following steps (i), (ii), or (iii):

(i) where

R¹ is --O--C(O)--R⁴, where R⁴ is independently selected from thosegroups recited for R⁴ above; and one of R^(1a) or R^(1b) is the same asR¹ and the other of R^(1a) or R^(1b) is hydroxyl;

the step of contacting said mixture I, in the presence of water and/oran organic alcohol, with an enzyme or microorganism capable ofcatalyzing the stereoselective hydrolysis of mixture I to provide saidmixture II; or

(ii) where

R¹ is hydroxyl; and

one of R^(1a) or R^(1b) is hydroxyl and the other of R^(1a)

or R^(1b) is R⁴ --C(O)--O--, where R⁴ is independently selected fromthose groups recited for R⁴ above;

the step of contacting said mixture I, in the presence of a compoundIII:

    R.sup.4 --C(O)--L                                          (III)

where R⁴ is as defined above for R^(1a) or R^(1b) and L is a leavinggroup, with an enzyme or microorganism capable of catalyzing thestereoselective esterification of mixture I to provide said mixture II;or

(iii) where

R¹ is a halogen atom; and

one of R^(1a) or R^(1b) is halogen and the other of R^(1a) or R^(1b) ishydroxyl;

the step of contacting said mixture I, in the presence of a hydroxideion donor, with an enzyme or microorganism capable of catalyzing thestereoselective dehalogenation of mixture I to provide said mixture II.

The above methods may be employed in the resolution of otherenantiomeric mixtures of the instant invention, although resolution ofthe above cis enantiomers Ia(1) and Ib(1) is preferred.

Preferred Methods for the Resolution of Mixture IV

Mixture IV is preferably resolved by stereoselective hydrolysis,esterification, dehalogenation or transesterification. A particularlypreferred method for the resolution of a mixture IV comprising theenantiamers IVa(1) and IVb(1): ##STR12## to form a mixture V comprisingcompounds Va(1) and Vb(1): ##STR13## where R² is heterocyclo orcycloalkyl;

R³ is hydrogen; R⁴ ; --C(O)--OR⁴ ; or --C(O)--R⁴,

where R⁴ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl orheterocyclo; and

R⁶ is hydrogen; or R⁴, where R⁴ is independently selected from thosegroups recited for R⁴ above;

comprises one of the following steps (i), (ii), or (iii):

(i) where

R¹ is --O--C(O)--R⁴, where R⁴ is independently selected from thosegroups recited for R⁴ above; and one of R^(1a) or R^(1b) is the same asR¹ and the other of R^(1a) or R^(1b) is hydroxyl;

the step of contacting said mixture IV, in the presence of water and/oran organic alcohol, with an enzyme or microorganism capable ofcatalyzing the stereoselective hydrolysis of mixture IV to provide saidmixture V; or

(ii) where

R¹ is hydroxyl; and

one of R^(1a) or R^(1b) is hydroxyl and the other of R^(1a) or R^(1b) isR⁴ --C(O)--O--, where R⁴ is independently selected from those groupsrecited for R⁴ above;

the step of contacting said mixture IV, in the presence of a compoundIII:

    R.sup.4 --C(O)--L                                          (III)

where R⁴ is as defined above for R^(1a) or R^(1b) and L is a leavinggroup, with an enzyme or microorganism capable of catalyzing thestereoselective esterification of mixture IV to provide said mixture V;or

(iii) where

R¹ is a halogen atom; and

one of R^(1a) or R^(1b) is halogen and the other of R^(1a) or R^(1b) ishydroxyl;

the step of contacting said mixture IV, in the presence of a hydroxideion donor, with an enzyme or microorganism capable of catalyzing thestereoselective dehalogenation of mixture IV to provide said mixture V.

A further particularly preferred method for the resolution of a mixtureIV comprising the enantiomers IVa(1) and IVb(1): ##STR14## to form amixture VI comprising compounds VIa(1) and VIb(1): ##STR15## where R¹ ishydroxyl; halo; or --O--C(O)--R⁴, where R⁴ is alkyl, alkenyl, alkynyl,aryl, cycloalkyl, cyloalkenyl or heterocyclo;

R² is heterocyclo or cycloalkyl; and

R³ is hydrogen; R⁴ ; --C(O)--OR⁴ ; or --C(O)--R⁴,

where R⁴ is independently selected from those groups recited for R⁴above;

comprises one of the following steps (i), (ii), or (iii):

(i) where

R⁶ is hydrogen; and

one of R^(6a) or R^(6b) is hydrogen and the other of R^(6a) or R^(6b) isR⁴, where R⁴ is independently selected from those groups recited for R⁴above;

the step of contacting said mixture IV, in the presence of an organicalcohol of the formula VII:

    R.sup.4 --OH                                               (VII)

where R⁴ is as defined above for R^(6a) or R^(6b), with an enzyme ormicroorganism capable of catalyzing the stereoselective esterificationof mixture IV to provide said mixture VI; or

(ii) where

R⁶ is R⁴, where R⁴ is independently selected from those groups recitedfor R⁴ above; and

one of R^(6a) or R^(6b) is the same as R⁶ and the other of R^(6a) orR^(6b) is hydrogen;

the step of contacting said mixture IV, in the presence of water, withan enzyme or microorganism capable of catalyzing the stereoselectivehydrolysis of mixture IV to provide said mixture VI; or

(iii) where

R⁶ is R⁴, where R⁴ is independently selected from those groups recitedfor R⁴ above; and

one of R^(6a) or R^(6b) is the same as R⁶ and the other of R^(6a) orR^(6b) is R⁷, where R⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl or heterocyclo, except that R⁷ is not the same as R⁶ ;

the step of contacting said mixture IV, in the presence of an organicalcohol of the formula VIII:

    R.sup.7 --OH                                               (VIII)

where R⁷ is as defined above, with an enzyme or microorganism capable ofcatalyzing the stereoselective transesterification of mixture IV toprovide said mixture VI.

The above methods may be employed in the resolution of otherenantiomeric mixtures of the instant invention, although resolution ofthe above enantiomers IVa(1) and IVb(1) is preferred.

The compound pairs so prepared, such as IIa(1) and IIb(1), arenon-enantiomeric and may subsequently be separated to yield opticallyactive, preferably optically pure, compounds. An optical purity greaterthan 99%, particularly 99.5%, is preferred.

The instant invention also provides a compound of the mixture I or IVsubstantially free of other isomers, which compound may be prepared bythe methods of the invention.

Definitions

The term "stereoselective conversion", as used herein, refers to thepreferential reaction of one enantiomer relative to another, that is,asymmetric, enantioselective, reaction. Likewise, the terms"stereoselective hydrolysis", "stereoselective esterification",stereoselective dehalogenation" and "stereoselectivetransesterification" refer to the preferential hydrolysis,esterification, dehalogenation and transesterification, respectively, ofone enantiomer relative to another.

The term "mixture", as said term is used herein in relation toenantiomeric compounds, denotes mixtures having equal (racemic) ornon-equal amounts of enantiomers.

The term "resolution" as used herein denotes partial, as well as,preferably, complete resolution.

The term "non-enantiomeric form" as used herein denotes the structure ofa compound, originally one of an enantiomeric pair, in which at leastone group has been modified so that said compound is no longer themirror image of the other compound of the original enantiomeric pair.

The terms "enzymatic process" or "enzymatic method" as used hereindenote a process or method of the present invention employing an enzymeor microorganism.

The terms "alkyl", "alkan" or "alk" as employed herein alone or as partof another group denote both straight and branched chain, optionallysubstituted hydrocarbons groups containing 1 to 15 carbons in the normalchain, preferably 1 to 6 carbons, such as methyl, ethyl, propyl,isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl,dodecyl, the various branched chain isomers thereof, and the like.Exemplary substituents include one or more groups selected from thefollowing: halo (especially chloro), trihalomethyl, alkoxy (for example,where two alkoxy substituents form an acetal), aryl such asunsubstituted aryl, alkyl-aryl or haloaryl, cycloalkyl such asunsubstituted cycloalkyl or alkyl-cycloalkyl, hydroxy or protectedhydroxy, carboxyl, alkyloxycarbonyl, alkylamino, alkylcarbonylamino,amino, arylcarbonylamino, nitro, cyano, thiol or alkylthio.

The term "alkenyl" as employed herein alone or as part of another groupdenotes such optionally substituted groups as described above for alkyl,further containing at least one carbon to carbon double bond. Exemplarysubstituents include one or more alkyl groups as described above, and/orone or more groups described above as alkyl substituents.

The term "alkynyl" as employed herein alone or as part of another groupdenotes such optionally substituted groups described above for alkyl,further containing at least one carbon to carbon triple bond. Exemplarysubstituents include one or more alkyl groups as described above, and/orone or more groups described above as alkyl substituents.

The term "cycloalkyl" as employed herein alone or as part of anothergroup denotes optionally substituted saturated cyclic hydrocarbon groupscontaining one to three rings and 3 to 12 ring carbons, preferably 3 to8 ring carbons, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, andadamantyl. Exemplary substituents include one or more alkyl groups asdescribed above, and/or one or more groups described above as alkylsubstituents.

The term "cycloalkenyl" as employed herein alone or as part of anothergroup denotes such optionally substituted groups as described above forcycloalkyl, further containing at least one carbon to carbon double bondin the ring system. Exemplary substituents include one or more alkylgroups as described above, and/or one or more groups described above asalkyl substituents.

The terms "aryl" or "ar" as employed herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Exemplarysubstituents (preferably three or fewer) include one or more of thefollowing groups: alkyl such as unsubstituted alkyl, haloalkyl, orcycloalkyl-alkyl, halogen, alkoxy such as unsubstituted alkoxy orhaloalkoxy, hydroxy, aryloxy such as phenoxy, R⁴ -carbonyloxy, where R⁴is as defined above, allyl, cycloalkyl, alkylamino, dialkylamino, amidosuch as alkylcarbonylamino or arylcarbonylamino, amino, nitro, cyano,alkenyl, thiol, R⁴ -carbonyl, where R⁴ is as defined above, ormethylenedioxy where the methylene group may be substituted by 1 or 2lower alkyl groups, 1 or 2 arylalkenyl groups, and/or 1 or 2 alkylthiogroups. Particularly preferred aryl groups are phenyl and substitutedphenyl, especially phenyl substituted by one or more hydroxyl, alkyland/or alkoxy groups.

The terms "halogen" or "halo" as used herein alone or as part of anothergroup refer to chlorine, bromine, fluorine, and iodine.

The terms "heterocyclo" or "heterocyclic" as used herein alone or aspart of another group denote optionally substituted fully saturated orunsaturated, monocyclic or bicyclic, aromatic or nonaromatic hydrocarbongroups having at least one heteroatom in at least one ring, andpreferably 5 or 6 atoms in each ring. The heterocyclo group preferablyhas 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogenatoms in the ring, and may be bonded to the remainder of the moleculethrough a carbon or heteroatom. Exemplary substituents include one ormore of the following groups: halogen, alkoxy, hydroxy, aryl such asphenyl or halophenyl, alkanoyloxy, arylcarbonyloxy such as benzoyloxy,alkyl such as aralkyl, alkylamino, alkanoylamino, arylcarbonylamino,amino, nitro, cyano, and thiol. Exemplary heterocyclo groups includethienyl, furyl, pyrrolyl, pyridyl; imidazolyl, pyrrolidinyl,piperidinyl, azepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl,benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, andbenzofurazanyl.

The term "hydroxyl protecting group" as used herein denotes a groupcapable of protecting a free hydroxyl group ("protected hydroxyl")which, subsequent to the reaction for which protection is employed, maybe removed without disturbing the remainder of the molecule. A varietyof protecting groups for the hydroxyl group and the synthesis thereofmay be found in "Protective Groups in Organic Synthesis" by T. W.Greene, John Wiley and Sons, 1981, or Fiser & Fiser. Exemplary hydroxylprotecting groups include methoxymethyl, 1-ethoxyethyl, benzyloxymethyl,(β-trimethylsilylethoxy)methyl, tetrahydropyranyl,2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl,trichloromethoxycarbonyl and 2,2,2-trichloroethoxymethyl.

Starting Materials

The starting materials for the present resolution methods may beobtained as described in the Examples herein, or by methods analogous tothose described in U.S. patent application Ser. No. 07/822,015, filedJan. 15, 1992.

The starting mixtures I or IV may contain, for example, thediastereomers of the compounds Ia and Ib or IVa and IVb, although it ispreferred that such compounds are separated prior to conducting theenzymatic resolution methods of the present invention.

Preferred Compounds

Cis compounds of the formula I have a stereoisomeric configuration whichis preferred in compounds employed as intermediates in the preparationof C-13 sidechain-bearing taxanes. Compounds of the mixtures I and IIhaving the same absolute stereoconfiguration corresponding to that of acompound Ia where R¹ is acetyloxy, R² is furyl and R³ is hydrogen in the3R,4S configuration are particularly preferred.

Erythro compounds of the formula IV have a stereoisomeric configurationwhich is preferred in compounds employed as intermediates in thepreparation of C-13 sidechain-bearing taxanes. Compounds of the mixturesIV, V and VI having the same absolute stereoconfiguration correspondingto that of a compound IVa(1) where R¹ is hydroxyl, R² is furyl, W is--NHR³ and R³ is hydrogen, and R⁶ is hydrogen in the 2R,3S configurationare preferred. In mixture IV, T^(a) = ##STR16## is preferred.

Resolution of β-lactams of the formula I is preferred.

In the compounds of the present invention, R¹ is preferably alkanoyloxy,such as unsubstituted alkanoyloxy (e.g., acetyloxy), or hydroxy; R² ispreferably furyl or thienyl; and R³ is preferably hydrogen, phenyl,substituted phenyl, phenylcarbonyl, substituted phenylcarbonyl,alkylcarbonyl, alkenylcarbonyl or alkoxycarbonyl such ast-butoxycarbonyl. R⁶ is preferably hydrogen or a C₁₋₆ alkyl such asmethyl.

Enzymes and Microorganisms

The enzyme or microorganism employed in the methods of the presentinvention may be any enzyme or microorganism having the ability tocatalyze the stereoselective conversions as described herein. Variousenzymes, such as esterases, lipases and proteases, regardless of originor purity, are suitable for use in the present invention. The enzymemay, for example, be in the form of animal or plant enzymes or mixturesthereof, cells of microorganisms, crushed cells, extracts of cells, orof synthetic origin.

With respect to the use of microorganisms, the methods of the presentinvention may be carried out using any microbial cellular materialhaving the ability to catalyze the stereoselective conversions asdescribed herein. The cells may be used in the form of intact wet cellsor dried cells such lyophilized, spray-dried or heat-dried cells. Cellsmay also be used in the form of treated cell material such as rupturedcells or cell extract.

The enzyme or microbial materials may be employed in the free state orimmobilized on a support (for example, a polymeric resin) such as byphysical adsorption or entrapment.

Exemplary genera of microorganisms suitable as sources of catalyzingenzymes include Mucor, Escherichia, Staphylococcus, Agrobacterium,Acinetobacter, Rhizopus, Aspergillus, Nocardia, Streptomyces,Trichoderma, Candida, Rhodotorula, Torulopsis, Proteus, Bacillus,Alcaligenes, Pseudomonas, Rhodococcus, Brevibacterium, Geotrichum,Enterobacter, Chromobacterium, Arthrobacter, Microbacterium,Mycobacterium, Saccharomyces, Penicillium, Methanobacterium, Botrytis,Chaetomium, Ophiobolus, Cladosporium and the like. The use ofgenetically engineered host cells is also contemplated.

Specific microorganisms suitable for use in the present processesinclude Chromobacterium viscosum, Pseudomonas aeuriginosa such as ATCC25619, Pseudomonas fluorescens, Pseudomonas putida such as ATCC 31303,Pseudomonas ovalis, Escherichia coli, Staphylococcus aureus, Alcaligenesfaecalis, Streptomyces griseus, Pseudomonas cepacia, Candida rugosa suchas ATCC 14830, Geotrichum candidum such as ATCC 32345, Streptomycesclavuligerus, Nocardia erthropolis, Nocardia asteraicdes, Mycobacteriumphlei, Agrobacterium radiobacter, Aspergillus niger, Rhizopus oryzae andthe like. Two or more, as well as a single, species of microorganism maybe employed when carrying out the instant processes.

The term "ATCC" as used herein refers to the accession number of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, the depository for the organism referred to.

The resolution methods of the instant invention may be carried outsubsequent to the growth of the microorganism(s) employed, orconcurrently therewith that is, in the latter case, by in situfermentation and resolution. The growth of microorganisms may beachieved by one of ordinary skill in the art, for example, by the use ofan appropriate medium containing nutrients such as carbon and nitrogensources and trace elements.

Exemplary, commercially available enzymes suitable for use in thepresent invention include lipases such as Amano PS-30 (Pseudomonascepacia), Amano GC-20 (Geotrichum candidum), Amano APF (Aspergillusniger), Amano AK (Pseudomonas sp.), Pseudomonas fluorescens lipase(Biocatalyst Ltd.), Amano Lipase P-30 (Pseudomonas sp.), Amano P(Pseudomonas fluorescens), Amano AY-30 (Candida cylindracea), Amano N(Rhizopus niveus), Amano R (Penicillium sp.), Amano FAP (Rhizopusoryzae), Amano AP-12 (Aspergillus niger), Amano MAP (Mucor meihei),Amano GC-4 (Geotrichum candidum), Sigma L-0382 and L-3126 (porcinepancrease), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde), KIDLipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma L-3001(Wheat germ), Sigma L-1754 (Candida cylindracea), Sigma L-0763(Chromobacterium viscosum) and Amano K-30 (Aspergillus niger).Additionally, exemplary enzymes derived from animal tissue includeesterase from pig liver, α-chymotrypsin and pancreatin from pancreassuch as Porcine Pancreatic Lipase (Sigma). Two or more, as well as asingle, enzyme may be employed when carrying out the instant processes.

The preferred embodiments of the instant invention are described furtherin the following Reaction Schemes. While, for clarity, these ReactionSchemes illustrate the resolution of certain cis enantiomeric mixtures,it is understood that the embodiments as described apply to theresolution of the other enantiomeric mixtures of the present inventionas well. ##STR17##

Mixtures I and IV may be stereoselectively esterified as illustrated inthe above Reaction Scheme I, and mixture IV may be stereoselectivelytransesterified as illustrated in the above Reaction Scheme II.

(A) Acylation

Mixture I may be selectively esterified to form mixture II, and mixtureIV may be selectively esterified to form mixture V by use of anacylating agent of the formula III:

    R.sup.4 --C(O)--L                                          (III).

In formula III, R⁴ may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl or heterocyclo group. Preferred R⁴ groups in formula IIIare alkyl groups such as C₁₋₆ alkyl groups, especially methyl. L is aleaving group which may be displaced to form an ester group. Exemplary Lgroups include halogen atoms, hydroxyl, alkoxy, or alkenyloxy groups.Preferred L groups are alkenyloxy groups, most preferably C₁₋₆alkenyloxy groups such as CH₂ ═CH--O-- and CH₂ ═C(CH₃)--O--. Anyacylation agent of formula III which effects esterification may beemployed, with isopropenyl acetate and vinyl acetate being particularlypreferred.

(B) Esterification with Alcohol

Mixture IV may be selectively esterified to form mixture VI by use of anorganic alcohol of the formula VII:

    R.sup.4 --OH                                               (VII).

In formula VII, R⁴ may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl or heterocyclo group. Alkyl groups, particularly C₁₋₆ alkylgroups, are preferred as R⁴.

(C) Transesterification with Alcohol

Mixture IV may be selectively transesterified to form mixture VI by useof an alcohol of the formula VIII:

    R.sup.7 --OH                                               (VIII).

In formula VIII, R⁷ may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl or heterocyclo group, except that R⁷ is not the same as R⁶.It is preferred that the group R⁷ be as distinct as possible from thegroup R⁶ to facilitate subsequent separation of the compound bearing thegroup R⁷ --O--C(O)-- from the compound bearing the group R⁶ --O--C(O)--.Thus, it is preferred to employ an alcohol of the formula VIII in whichthe R⁷ group differs with respect to the group R⁶ in terms of molecularweight, or otherwise imparts distinctive physical or chemical propertiesto the transesterified ester.

The esterification (acylation) procedure (A), and the esterification andtransesterification procedures (B) and (C), are preferably carried outin an organic solvent. Exemplary solvents suitable for use in theseprocesses include 1,1,2-trichloro-1,2,2-trifluoroethane, toluene,cyclohexane, benzene, hexane, heptane, isooctane, octane, methyl ethylketone, methyl isobutyl ketone and the like. Water is preferably addedto the reaction mixture in small amounts. When present, theconcentration of water in the reaction mixture is preferably from about0.01% to about 1% based on the weight of solvent, or present in aconcentration less than or equal to that where the organic solvent issaturated. Water is most preferably present in an amount of about 0.05%to about 0.5% based on the weight of solvent. The reaction solutionpreferably contains between about 5 to about 250 mg of the enantiomericstarting compounds per ml of solvent.

To carry out these processes, a compound III, VII or VIII is added tothe reaction medium. Preferred molar ratios of the compound III:compounds of mixture I or IV are from about 1:1 to about 4:1; preferredmolar ratios of the compound VII: compounds of mixture IV are from about1:1 to about 4:1; and preferred molar ratios of the compound VIII:compounds of mixture IV are from about 1:1 to about 4:1.

The enzymes or microorganisms employed in these procedures arepreferably lipases or esterases or microorganisms capable of producingthese enzymes. Enzymes or microorganisms particularly preferred in theseprocesses are Lipase PS-30 from Pseudomonas sp., Lipase P-30 fromPseudomonas sp., Lipase R from Penicillium sp., Lipase OF, Lipase N fromRhizopus niveus, Lipase APF from Aspergillus niger, Lipase GC-20 fromGeotrichum candidum, Lipase AK from Pseudomonas sp., Lipase AY-30 fromCandida sp., and Pseudomonas fluorescens Lipase.

An enzyme may, for example, be used in its free state or in immobilizedform. A preferred embodiment of the invention is that where an enzyme isadsorbed onto a suitable carrier, e.g., diatomaceous earth (porousCelite Hyflo Supercel), microporous polypropylene (Enka Accurel®polypropylene powder), or a nonionic polymeric adsorbent such asAmberlite® XAD-2 (polystyrene) or XAD-7 (polyacrylate) from Rohm andHaas Co. When employed to immobilize an enzyme, a carrier may controlthe enzyme particle size and prevent aggregation of the enzyme particleswhen used in an organic solvent. Immobilization can be accomplished, forexample, by precipitating an aqueous solution of the enzyme with coldacetone in the presence of the Celite Hyflo Supercel followed by vacuumdrying, or in the case of a nonionic polymeric adsorbent, incubatingenzyme solutions with adsorbent on a shaker, removing excess solutionand drying enzyme-adsorbent resins under vacuum. The enzyme ispreferably added to the reaction solution to achieve concentrationsranging from about 5 to about 200 mg of enzyme per ml of solvent. Whileit is desirable to use the least amount of enzyme possible, the amountof enzyme required will vary depending upon the specific activity of theenzyme used.

These processes may also be carried out using microbial cells containingan enzyme having the ability to catalyze the stereoselectiveconversions. When using a microorganism to perform the resolution, theseprocedures are conveniently carried out by adding the cells and theenantiomeric mixture starting material to the desired reaction medium.Cells may be used in the form of intact cells, dried cells such aslyophilized, spray-dried or heat-dried cells, immobilized cells, orcells treated with organic solvents such as acetone or toluene. Cellsmay also be used in the form of treated cell material such as rupturedcells or cell extract. Cell extracts immobilized on Celite® or Accurel®polypropylene as described earlier may also be employed.

Incubation of the reaction medium is preferably at a temperature betweenabout 4 and about 60° C. and is most preferably between about 30° to 50°C. The reaction time can be appropriately varied depending upon theamount of enzyme used and its specific activity. Reaction times may bereduced by increasing the reaction temperature and/or increasing theamount of enzyme added to the reaction solution. ##STR18##

As can be seen from Reaction Scheme III above, mixtures I and IV may bestereoselectively hydrolyzed to form mixtures II and V, respectively, byuse of water and/or an organic alcohol, and mixture IV may bestereoselectively hydrolyzed to form mixture VI by use of water. Thegroups R⁴, forming part of R¹, and R⁶ in the starting enantiomericcompounds are preferably alkyl, most preferably C₁₋₆ alkyl such asmethyl.

A compound of the formula IX:

    R.sup.8 --OH                                               (IX)

may be employed as the organic alcohol, where R⁸ is an alkyl, alkenyl,alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo group, and ispreferably alkyl such as methyl. Use of the organic alcohol IX mayresult in the formation of the by-product ester R⁴ --C(O)--OR⁸. Use ofwater as the hydrolysis agent may result in the formation of theby-product acid R⁴ --C(O)--OH. To maintain a steady pH as these acidicby-products are generated, a base such as an alkali metal hydroxide maybe added. When an organic alcohol IX is employed, an amount providing amolar ratio of compound IX: compounds of mixtures I or IV of from about1:1 to about 4:1 is preferably added.

These processes preferably employ water-soluble enzymes capable ofcatalyzing stereoselective hydrolysis. Especially suitable for use withthese processes are lipases and esterases, as well as pancreatin andα-chymotrypsin. Either the crude or purified forms of these enzymes, infree form or immobilized on a support (for example, on a resin such asXAD-7, XAD-2 or Accurel® resins), may be employed. Particularlypreferred in these processes are Lipase PS-30 from Pseudomonas sp.(Pseudomonas cepacia) (Amano Int'l), Lipase P-30 (Amano) fromPseudomonas sp., Lipase GC-20 Geotrichum candidum (Amano Int'l), LipaseN Rhizopus niveus (Amano Int'l), Lipase APF Aspergillus niger (AmanoInt'l), Lipase AY-30 Candida sp. (Amano), Lipase AK Pseudomonas sp.(Amano Int'l), Pseudomonas fluorescens Lipase (Biocatalyst Ltd.),Esterase 30,000 (Gist-Brocarde), Lipase OF (Sepracor), KID Lipase(Gist-Brocarde), Lipase R (Rhizopus sp., Amano Int.) and PorcinePancreatic Lipase (Sigma Chem).

The above hydrolyses are preferably conducted in an aqueous, such as abuffered aqueous (e.g., phosphate buffer), medium or in an aqueousmedium containing a miscible or immiscible organic solvent. For example,the reaction may be conducted in a biphasic solvent system comprising anorganic phase, immiscible in water, and an aqueous phase. Use of a twophase solvent system may enhance the efficiency of such processes wherethe substrate material is insoluble in water.

Solvents for the organic phase of a biphasic solvent system may be anyorganic solvent immiscible in water, such as toluene, cyclohexane,xylene, trichlorotrifluoroethane and the like. The aqueous phase isconveniently of water, preferably deionized water, or a suitable aqueousbuffer solution, especially a phosphate buffer solution. The biphasicsolvent system preferably comprises between about 10 to 90 percent byvolume of organic phase and between about 90 to 10 percent by volume ofaqueous phase.

An amount of enantiomeric mixture starting material of from about 0.1 toabout 100 mg per ml of reaction solution, and one or more enzymes in anamount of from about 0.1 to about 100 mg enzyme per mg of startingmaterial to be hydrolyzed, is preferred.

The reaction mixture is preferably adjusted to and maintained at aboutpH 7.0, preferably with an aqueous alkali metal hydroxide, carbonate orbicarbonate.

The reaction time may be selected based on the enzyme, the temperatureand the enzyme concentration. Temperatures of from about 4° C. to about60° C. are preferably employed. ##STR19##

As can be seen from Reaction Scheme IV above, mixtures I and IV may beselectively dehalogenated to form mixtures II and V, respectively,wherein X denotes a halogen atom.

Any compound capable of effecting these reactions may be employed as thehydroxide ion donor. Exemplary such compounds are selected from water,alkali or alkaline earth metal hydroxides such as sodium and potassiumhydroxide, and ammonium hydroxides such as quaternary ammoniumhydroxides, for example, those of the formula (R⁹)₄ NOH where R⁹ ishydrogen or alkyl, particularly potassium hydroxide and water. Amountsof the hydroxide ion donor added are preferably those providing a molarratio of hydroxide ion donor: mixture I or IV enantiomeric startingmaterial of from about 1:1 to about 4:1.

A reaction medium containing water and an organic solvent such astoluene or hexane is preferably employed. The enantiomeric startingmaterials are preferably employed in an amount of from about 1 mg toabout 100 mg per ml of solvent.

Enzymes or microorganisms employed in the dehalogenation reaction arepreferably selected from the genera Pseudomonas, Trichoderma,Acinetobacter, Alcaligenes, Nocardia, Mycobacterium, Rhodococcus,Methanobacterium, Proteus, or enzymes derived therefrom, and arepreferably employed in amounts of from about 0.1 mg to about 10 mgenzyme per mg of starting material to be dehalogenated.

Temperatures of from about 4° C. to about 50° C. are preferablyemployed.

Separation

The products of the stereoselective conversions may be isolated andpurified by methodologies such as extraction, distillation,crystallization, column chromatography, and the like.

A preferred method for separating the product mixtures formed by themethods of the present invention is by liquid-liquid extraction.

Utility

Taxanes are diterpene compounds containing the taxane carbon skeleton:##STR20## which skeleton may contain ethylenic unsaturation in the ringsystem thereof. Of particular interest are taxanes having the abovecarbon skeleton wherein the 11,12-positions are bonded through anethylenic linkage, and the 13-position contains a side chain.Pharmacologically active taxanes, such as taxol analogues, may be usedas antitumor agents to treat patients suffering from cancers such asovarian cancer, melanoma, breast, colon or lung cancer, and leukemia.

The resolved compounds obtained by the methods of the present inventionare particularly useful as intermediates in forming the aforementionedC-13 side chain on the taxane skeleton. The addition of such a sidechain, in and of itself, may impart an increased or more desirablepharmacological activity to the taxane product, or may form a taxaneproduct which is more readily converted to a taxane having an increasedor more desirable pharmacological activity than the starting compound.

The compounds resolved according to the methods of the present inventionmay be modified prior to use in side chain formation. For example,resolved compounds containing an azide group N₃ as the group W may betreated by a reducing agent to form an amine group which may besubstituted.

Exemplary methods for side chain formation, and taxane products whichmay be formed employing such methods, include those described inEuropean Patent Application No. 534,708; U.S. patent application Ser.No. 08/080,704, filed Jun. 28, 1993; U.S. patent application Ser. No.08/029,819, filed Mar. 11, 1993; U.S. patent application Ser. No.07/996,455, filed Dec. 24, 1992; U.S. patent application Ser. No.08/062,687, filed May 20, 1993; U.S. patent application Ser. No.07/981,151, filed Nov. 24, 1992; and U.S. patent application Ser. No.07/995,443, filed Dec. 23, 1992; all of which aforementioned documentsare incorporated herein by reference.

Salts or solvates of reactants or products may be employed or preparedas appropriate or desired in the methods of the present invention.

The methods of the present invention are further described by thefollowing examples. These examples are illustrative only, and are in noway intended to limit the scope of the instant claims.

EXAMPLE 1 Synthesis of Substrate for Resolution(±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one

(A) Preparation of N,N'- (2'-Furanyl)methyl!-2-furanmethanimine##STR21##

To a 3 L 2-necked round bottom flask equipped with a thermometer andmechanical stirrer was added 2-furaldehyde (furfural, 311 ml, 3.75 mol,Aldrich) and 2-propanol (IPA, 0.75 L, reagent grade). With stirring, 1.5L of concentrated NH₄ OH (aq., ˜30%) (22.2 mol) was added in oneportion. An exotherm (˜35° C.) was noted in the first two hours. Theresulting beige powder was isolated by filtration (Whatman No. 1),washed with water (1.5 L) and dried overnight in vacuo at 30° C. Thisgave 255.3 g (76.1% yield) of the title hydrofuramide as a beige powder(melting point (mp)=116°-117° C.). The R_(f) of the title hydrofuramidewas 0.5 (ethyl acetate (EtOAc)/Hexane, 1:1; UV visualization).

(B) Preparation of (±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one

(i) Staudinger Reaction ##STR22## "Ac" denotes acetyl (CH₃ --C(O)--)"(±)" denotes, with respect to the 3-and 4-position substituents on theazetidine ring, a racemate of the cis enantiomers.

To a 2 L 3-necked round bottom flask equipped with a thermometer,pressure equalizing dropping funnel and mechanical stirrer was added thehydrofuramide title product of step (A) above (80.48 g, 0.300 mol) andethyl acetate (1.0 L, reagent grade). This mixture was cooled underargon to 4° C. at which point triethylamine (50.2 ml, 0.360 mol,Aldrich) was added in one portion. The dropping funnel was charged withacetoxyacetyl chloride (37.0 ml, 0.341 mol, Aldrich) and ethyl acetate(0.50 L, reagent grade) and this solution was added dropwise over aperiod of 1 hour. After an additional 2 hours, the stirring wasdiscontinued and the reaction vessel sealed (Parafilm "M"®) and moved tothe cold room (4° C.) for a further 15 hours. The heterogeneous reactionmixture was allowed to warm to 22° C. (˜1 hour) with stirring andtransferred to a 4.0 L separatory funnel and washed with aqueous NH₄ Cl(sat) (500 ml). Both layers were filtered through glass microfibrefilter paper (Whatman) to remove a fine, black suspension. Removal ofthe particulate material aided in phase separation. The filter cake wasrinsed with ethyl acetate (50 ml) and the filtrate transferred back tothe separatory funnel and the aqueous layer (pH=6.3) removed. Theorganic layer was then washed with another portion of aqueous NH₄ Cl(sat) (250 ml; pH=5.9), aqueous NaHCO₃ (sat) (400 ml; pH=8.6) andaqueous NaCl (sat) (400 ml; pH=7.0),. The organic layer was filteredthrough glass microfibre filter paper (Whatman) and divided into 2 equalportions (750 ml each). These solutions were used directly in the nextstep.

(ii) Deprotection ##STR23##

To two 2.0 L Parr flasks each containing 10% palladium on activatedcarbon (2×6.00 g, Aldrich) was added, under a stream of argon, theorganic layers from above (2×750 ml; Step (B)(i)). This mixture wastreated with hydrogen (4 atm) for 1 day at ambient temperature. Thecatalyst was removed by filtration through a pad of Celite® and thefilter cake rinsed with ethyl acetate (100 ml). The filtrate wastransferred to a 4.0 L separatory funnel and washed twice with 1N HCl(500 ml, 250 ml; pH=0.76). The aqueous washings were combined andre-extracted with ethyl acetate (500 ml) and the organic layers werecombined and washed with aqueous NaHCO₃ (sat) (400 ml; pH=8.34) andaqueous NaCl (sat) (400 ml; pH=7.5). The organic layer was dried overMgSO₄ (˜100 g) and treated with activated decolorizing charcoal (30 g,BDH). After 15 minutes, the mixture was filtered through a pad ofCelite® and concentrated in vacuo to 160 ml, cooled overnight (4° C.)and the precipitated solid isolated by filtration through filter paper(Whatman No. 1). The filter cake was rinsed with diethyl ether andhexane (100 ml of each) to provide, after drying in vacuo, 35.98 g(61.4% yield from the above hydrofuramide) of the title product(±)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one as white needles (mp118°-119° C.). HPLC quantitative analysis demonstrated 8.10 g of thetitle product in the mother liquor. The activity yield, from the abovehydrofuramide, was therefore 75.3%.

EXAMPLE 2 Stereoselective Hydrolysis of(±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one

The substrate employed in this Example was the racemate:

I ##STR24## prepared as the title product of Example 1. The products ofthe stereoselective hydrolysis of this Example were the compounds:

(+)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one: ##STR25##(-)-cis-3-Hydroxy-4-(2'-furanyl)azetidin-2-one: ##STR26##

The stereoselective hydrolysis was conducted as follows. A reactionmixture in 1 L of 25 mM potassium phosphate buffer pH 7.0 was preparedcontaining 10 grams of substrate and 100 grams of lipase PS-30 fromPseudomonas sp. (Amano International Co.). The reaction was carried outat 30° C., 150 revolutions-per-minute (RPM) agitation. During thereaction, the pH of the reaction mixture was maintained at 7.0 with 5NNaOH using a pH stat. The hydrolysis reaction was monitored by highpressure liquid chromatography (HPLC). Periodically, samples (1 ml) weretaken and extracted with 10 ml of ethyl acetate. The ethyl acetate layerwas separated and evaporated to dryness and analyzed by HPLC (asdescribed following) for the substrate and product concentrations andthe optical purity of the product. The results obtained are as shown inthe following Table 1.

                  TABLE 1    ______________________________________                                     Optical Purity    Reaction Time             Conversion  Yield       of Product    (Hours)  (% Product IIb)                         (% Product IIa)                                     IIa (%)    ______________________________________    4        18          82          --    8        34          66          --    12       46          54          --    16       51          49          >99.4    ______________________________________

In Process Chiral HPLC Assays

Periodically, during the reaction as described above, a 1 ml sample wastaken and extracted with 10 ml of ethyl acetate contained in a 50 mlscrew-cap tube. The ethyl acetate layer (5 ml) was removed, evaporatedunder a gentle stream of nitrogen. The residue was dissolved in 2 ml ofmobile phase (hexane:absolute ethanol, 95:5). The mobile phase waspassed through 0.2 μm Lydex filter and about 1 ml of the filtrate wastransferred to a crimp vial for HPLC analysis. HPLC conditions:

Hewlett Packard 1090 chromatogram.

Column: Chiralcal AD

Mobile Phase: hexane:absolute ethanol, 95:5

Column Temperature: Ambient

Flow Rate: 1 ml/min

Detection: 210 nm.

The retention times for the two enantiomers of racemic acetate were 23.2min and 28.9 min, respectively. The retention times for the twoenantiomers of racemic alcohol were 63.9 min and 74.8 min, respectively.

EXAMPLE 3 Stereoselective Hydrolysis of(±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one using Immobilized LipasePS-30 Enzyme

The substrate employed, and the products obtained, were those of Example2 above.

Immobilization of Enzyme

Three different carriers--XAD-7 (Amberlite XAD-7 nonionic polymericadsorbent, 20-60 mesh polyacrylate resin), XAD-2 (Amberlite XAD-nonionicpolymeric adsorbent, 20-60 mesh polystyrene resin) and Accurel PP(polypropylene resin 200-400 microns)--were used for the immobilizationprocedures.

Crude Amano PS-30 lipase (10 g) was dissolved in 25 ml of distilledwater and centrifuged at 10,000 RPM for 10 minutes to obtain clearsupernatant. The carrier (1.3 g) in a 25 ml vial was washed 5 times withmethanol and added to enzyme solution in a flask and gently agitated ona gyrotory shaker at room temperature. Adsorption of enzyme to thecarrier was checked periodically by lipase assay (Sigma olive oilemulsion as substrate) and by protein remaining in filtrate. About 68%,71% and 98% adsorption efficiencies were obtained using XAD-7, XAD-2 andAccurel resins, respectively. After complete immobilization (20 to 24hours), the carrier-enzyme slurry was filtered through a milliporefilter and the carrier was washed with about 300 ml of distilled water.Subsequently, the carrier containing the immobilized lipase was dried ina vacuum oven at room temperature.

Use of Immobilized Enzyme

Immobilized enzyme was employed for the enzymatic hydrolysis reactiondescribed in Example 2. Reaction mixtures were prepared which contained30 ml of 25 mM potassium phosphate buffer pH 7.0 containing 300 mg ofsubstrate as described in Example 2, and 300 mg of the above preparedimmobilized Lipase PS-30. The reactions were conducted as described inExample 2. The results obtained are shown in the following Table 2.

                  TABLE 2    ______________________________________                                         Optical            Reaction                     Purity of    Immobilized            Time     Conversion Yield    Product IIa    Support (Hours)  (% Product IIb)                                (% Product IIa)                                         (%)    ______________________________________    XAD-2   3        53         47       99.0    XAD-7   3        54         46       99.3    Accurel PP            3        51         49       99.5    ______________________________________

EXAMPLE 4 Stereoselective Hydrolysis of(±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one

The substrate employed, and products obtained, were those of Example 2above. In this Example, a number of reactions were run in which lipasesfrom different sources were employed.

In each reaction, the reaction mixture, in 20 ml of 25 mM phosphatebuffer, pH 7.0 contained 1 gram of crude lipase and 200 mg of substrate.The reactions were conducted at 25° C. in a pH stat at pH 7.0. Theresults obtained are shown in the following Table 3.

                  TABLE 3    ______________________________________                              Reaction Time    Enzyme        Source      (Hours)    ______________________________________    Lipase PS-30  Amano Int.  16    Pseudomonas sp.    Lipase AY-30  Amano Int.   8    Candida sp.    Lipase AK     Amano Int.  16    Pseudomonas sp.    Pseudomonas   Biocatalyst Ltd.                              16    fluorescens    Porcine pancreatic                  Sigma       19    Lipase    Esterase 30,000                  Gist-Brocarde                              24    Lipase OF     Sepracor     8    KID Lipase    Gist-Brocarde                              48    Lipase R.     Amano Int.  24    Rhizopus sp.    ______________________________________    Conversion   Yield      Optical Purity of    (% Product IIb)                 (% Product IIa)                            Product IIa (%)    ______________________________________    52           48         >99.5    55           45         99.0    53           47         99.3    55           45         98.8    52           48         99.8    51           49         95    56           44         99.5    52           48         95    56           44         99.0    ______________________________________

EXAMPLE 5 Stereoselective Acetylation (Esterification) of(±)-cis-3-Hydroxy-4-(2'-furanyl)azetidin-2-one

The substrate employed in this Example was the racemate: ##STR27##prepared by chemical hydrolysis (using Na₂ CO₃) of the correspondingracemic acetate. The products of the stereoselective acetylation of thisExample were the compounds:

(+)-cis-3-hydroxy-4-(2'-furanyl)azetidin-2-one: ##STR28##(-)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one: ##STR29##

In this Example, a number of reactions were run in which lipases fromdifferent sources were employed to achieve stereoselective acetylation.In each reaction, the reaction mixture, in 25 ml of toluene, contained 1gram of crude lipase and 100 mg of substrate, 800 mg of isopropenylacetate, and 0.05% water. The reactions were conducted at 30° C. and 100RPM on a shaker. The products and substrate were analyzed by HPLC. Theresults which were obtained are shown in the following Table 4.

                  TABLE 4    ______________________________________                                          Optical                         Conversion                                   Yield (%                                          Purity of                         (% Product                                   Product                                          Product    Enzyme   Source      IIb)      IIa)   IIa (%)    ______________________________________    Lipase PS-30             Amano Int.  52        48     99.5    Lipase AY-30             Amano Int.  55        45     99.0    Lipase R Amano Int.  51        49     96.8    Pseudomonas             Biocatalyst Inc.                         54        46     98.8    fluorescens    Lipase OF             Sepracor    52        48     99.0    Porcine Pan-             Sigma       54        46     98.0    creatic Lipase    ______________________________________

EXAMPLE 6 Stereoselective Hydrolysis: Evaluation of Various Enzymes

The substrate employed, and products obtained, were those of Example 2above. For each stereoselective hydrolysis reaction, a reaction mixturein 20 ml of 25 mM potassium phosphate buffer pH 7.0 was preparedcontaining 200 mg of substrate and 1 gram of enzyme (see Table 5 forenzyme). The reaction was carried out at 30° C., 150revolutions-per-minute (RPM) agitation. During the reaction, the pH ofthe reaction mixture was maintained at 7.0 with 1N NaOH using a pH stat.The hydrolysis reaction was monitored by high pressure liquidchromatography. Periodically, samples (1 ml) were taken and extractedwith 4 ml of ethyl acetate. The ethyl acetate layer was separated andevaporated to dryness and analyzed by HPLC for the substrate and productconcentrations and the optical purity of the product. The resultsobtained are shown in the following Table 5.

                  TABLE 5    ______________________________________                                          Optical                                   Yield (%                                          Purity of                        Reaction Time                                   Product                                          Product    Enzyme   Source     (Hours)    IIa)   IIa (%)    ______________________________________    Lipase PS-30             Amano Int. 16         48     >99.5    Lipase AY-30             Amano Int.  8         46     >99    Lipase AK             Amano Int. 16         47     >99    Pseudomonas             Biocatalysts                        16         45     >99    fluorescens             Ltd.    Lipase    Porcina Pan-             Sigma      19         47     98.8    creatic Lipase    Esterase Gist/Brocarde                        24         35     99    30,000    Lipase OF             Sepracor    8         44     99    KID Lipase             Gist/Brocarde                        48         38     95    Lipase R Amano Int. 24         44     99    ______________________________________

EXAMPLE 7 Kinetics of Resolution of(±)-cis-3-Acetoxy-4-(2'-furanyl)azetidin-2-one by Immobilized LipasePS-30

The substrate employed, and products obtained, were the same as those ofExample 2 above.

A reaction mixture in 8.5 L of 25 mM potassium phosphate buffer pH 7.0was prepared containing 85 grams of substrate, and 85 grams of LipasePS-30 from Pseudomonas sp. (Amano International Co.). Lipase PS-30 wasimmobilized on Accural polypropylene and used in the reaction mixture.The reaction was carried out at 30° C., 150 revolutions-per-minute (RPM)agitation. During the reaction, the pH of the reaction mixture wasmaintained at 7.0 with 5N NaOH using a pH stat. The hydrolysis reactionwas monitored by high pressure liquid chromatography. Periodically,samples (1 ml) were taken and extracted with 4 ml of ethyl acetate. Theethyl acetate layer was separated and evaporated to dryness and analyzedby HPLC for the substrate and product concentrations and the opticalpurity of the product. The results which were obtained are shown in thefollowing Table 6.

                  TABLE 6    ______________________________________    Reaction Time              A-Acetate B-Acetate   A-Alcohol    (Hours)   (mg/ml)   (mg/ml)     (mg/ml)    ______________________________________    0         6.2       5.2         0    1         2.1       5.2         2.9    2         0.65      5.2         4.1    3         0.19      5.1         4.3    4         0.1       5           4.9    5         trace     4.95        4.9    6         trace     4.9         4.9    ______________________________________    Reaction Time              B-Alcohol Optical Purity                                    Optical Purity    (Hours)   (mg/ml)   B-Acetate (%)                                    A-Alcohol (%)    ______________________________________    0         0         50          >99    1         0         72          >99    2         0         89.6        >99    3         trace     98.5        >99    4         trace     98.5        >99    5         0.13      >99         97    6         0.15      >99         97    ______________________________________     ##STR30##     ##STR31##     ##STR32##     ##STR33##

EXAMPLE 8 Stereoselective Esterification of(±)-cis-3-Hydroxy-4-(2'-furanyl)azetidin-2-one

In this Example, the substrate employed, and products obtained, were thesame as those of Example 5. A reaction mixture in 10 ml of methyl ethylketone (MEK) was prepared containing 20 grams of substrate, 1 gram ofenzyme (see Table 7) and 0.4 ml isopropenyl acetate as acyl donor. Thereaction was carried out at 30° C., 150 revolutions-per-minute (RPM)agitation. The esterification reaction was monitored by high pressureliquid chromatography (described following) to determine substrate andproduct concentrations and the optical purity of the product. Theresults obtained are as shown in the following Table 7.

                  TABLE 7    ______________________________________                                    Yield of                         Reaction Time                                    Chiral    Enzyme   Source      (Hours)    acetate (%)    ______________________________________    Lipase PS-30             Amano Int.  96         50.5    Lipase AY-30             Amano Int.  120        51    Lipase AK             Amano Int.  120        49    Lipase OF             Sepracor    96         49.5    ______________________________________                       Optical   Yield of                                         Optical                       Purity of Chiral  Purity of    Enzyme   Source    acetate (%)                                 alcohol (%)                                         alcohol (%)    ______________________________________    Lipase PS-30             Amano Int.                       99.5      49.5    99    Lipase AY-30             Amano Int.                       99        49      99    Lipase AK             Amano Int.                       99.5      51      99    Lipase OF             Sepracor  99.4      50.5    99    ______________________________________

HPLC Method

The substrate and products were analyzed in the above Example by HPLC. ANova Pak C18 reverse phase column (3.9×150 mm) column was used. Themobile phase was 15% acetonitrile in water and the flow rate was 1ml/min. The detection wavelength was 227 nm. The retention times foralcohol and acetate were 2.7 min and 14.9 min, respectively.

The optical purity of chiral acetate was determined by chiral HPLC. AChiralpak AS column was used. The mobile phase consisted ofhexane:ethanol (96:4) which was used at 1 ml/min at ambient temperature.The detection wavelength was 210 nm. The retention times for the twoenantiomers of racemic acetate were 23.7 min and 20.9 min, respectively.The retention times for the two enantiomers of racemic alcohol were 63.9min and 74.8 min, respectively.

What is claimed is:
 1. A method for the resolution of a mixture Icomprising the enantiomers Ia and Ib, where R¹ is in the cis positionrelative to R² in both Ia and Ib, or where R¹ is in the trans positionrelative to R² in both Ia and Ib: ##STR34## to form a mixture IIcomprising the compounds IIa and IIb, where R^(1a) and R^(1b) are in thecis position relative to R² in both IIa and IIb, or where R^(1a) andR^(1b) are in the trans position relative to R² in both IIa and IIb, ineach case corresponding to the position of R¹ in mixture I: ##STR35##where R¹ is --O--C(O)--R⁴, and R⁴ is alkyl or alkenyl;one of R^(1a) orR^(1b) is the same as R¹ and the other of R^(1a) or R^(1b) is hydroxyl;R² is furyl or thienyl; and R³ is hydrogen or aryl;comprising the stepof contacting said mixture I, in the presence of water and/or an organicalcohol, with an enzyme which is selected from the group consisting oflipases derived from Pseudomonas, Candida, and Rhizopus, and porcinepancreatic lipase, and which is capable of catalyzing thestereoselective hydrolysis of said mixture I to provide said mixture II,and effecting said conversion.
 2. The method of claim 1, wherein amixture I comprising the enantiomers Ia(1) and Ib(1) is resolved:##STR36## to form a mixture II comprising the compounds IIa(1) andIIb(1): ##STR37##
 3. The method of claim 2, wherein, in said mixture Icomprising Ia(1) and Ib(1):R¹ is alkanoyloxy; R² is furyl or thienyl;and R³ is hydrogen, phenyl or substituted phenyl.
 4. The method of claim3, wherein said mixture I comprises(±)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one and said mixture IIcomprises (+)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one and(-)-cis-3-hydroxy-4-(2'-furanyl)azetidin-2-one.
 5. The method of claim1, wherein said enzyme is immobilized on a support.
 6. The method ofclaim 1, wherein, further, the non-enantiomeric compounds IIa and IIb ofsaid mixture II obtained are separated by a separation step.
 7. Themethod of claim 6, wherein said separation step is an extraction,distillation, crystallization, or column chromatography step.
 8. Themethod of claim 1, comprising the further step of preparing a taxanebearing a C-13 sidechain, wherein said sidechain is formed using atleast one of the non-enantiomeric compounds IIa or IIb of said mixtureII.
 9. A method for the resolution of a mixture I comprising theenantiomers Ia and Ib, where R¹ is in the cis position relative to R² inboth Ia and Ib, or where R¹ is in the trans position relative to R² inboth Ia and Ib: ##STR38## to form a mixture II comprising the compoundsIIa and IIb, where R^(1a) and R^(1b) are in the cis position relative toR² in both IIa and IIb, or where R^(1a) and R^(1b) are in the transposition relative to R² in both IIa and IIb, in each case correspondingto the position of R¹ in mixture I: ##STR39## where R¹ is hydroxyl;oneof R^(1a) or R^(1b) is hydroxyl and the other of R^(1a) or R^(1b) is R⁴--C(O)--O--, and R⁴ is alkyl or alkenyl; R² is furyl or thienyl; and R³is hydrogen or aryl;comprising the step of contacting said mixture I, inthe presence of a compound III:

    R.sup.4 --C(O)--L                                          (III)

where R⁴ is as defined above for R^(1a) or R^(1b) and L is a leavinggroup, with an enzyme which is selected from the group consisting oflipases derived from Pseudomonas, Candida, and Rhizopus, and porcinepancreatic lipase, and which is capable of catalyzing thestereoselective esterification of said mixture I to provide said mixtureII, and effecting said conversion.
 10. The method of claim 9, wherein amixture I comprising the enantiomers Ia(1) and Ib(1) is resolved:##STR40## to form a mixture II comprising the compounds IIa(1) andIIb(1): ##STR41##
 11. The method of claim 10, wherein, in said mixtureII comprising IIa(1) and IIb(1):R^(1a) or R^(1b) is alkanoyloxy; R² isfuryl or thienyl; and R³ is hydrogen, phenyl or substituted phenyl. 12.The method of claim 11, wherein said mixture I comprises(±)-cis-3-hydroxy-4-(2'-furanyl)azetidin-2-one, said mixture IIcomprises (+)-cis-hydroxy-4-(2'-furanyl)azetidin-2-one and(-)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one, and said compound of theformula III is isopropenyl acetate.
 13. The method of claim 9, whereinsaid enzyme is immobilized on a support.
 14. The method of claim 9,wherein, further, the non-enantiomeric compounds IIa and IIb of saidmixture II obtained are separated by a separation step.
 15. The methodof claim 14, wherein said separation step is an extraction,distillation, crystallization, or column chromatography step.
 16. Themethod of claim 9, comprising the further step of preparing a taxanebearing a C-13 sidechain, wherein said sidechain is formed using atleast one of the non-enantiomeric compounds IIa or IIb of said mixtureII.
 17. A method for the resolution of a mixture I comprising theenantiomers Ia and Ib, where R¹ is in the cis position relative to R² inboth Ia and Ib, or where R¹ is in the trans position relative to R² inboth Ia and Ib: ##STR42## to form a mixture II comprising the compoundsIIa and IIb, where R^(1a) and R^(1b) are in the cis position relative toR² in both IIa and IIb, or where R^(1a) and R^(1b) are in the transposition relative to R⁴ in both IIa and IIb, in each case correspondingto the position of R¹ in mixture I: ##STR43## where R¹ is --O--C(O)--R⁴,and R⁴ is alkyl or alkenyl;one of R^(1a) or R^(1b) is the same as R¹ andthe other of R^(1a) or R^(1b) is hydroxyl; R² is furyl or thienyl; andR³ is hydrogen or aryl;comprising the step of contacting said mixture I,in the presence of water and/or an organic alcohol, with an enzyme whichis selected from the group consisting of KID lipase, lipase OF andEsterase 30,000, and which is capable of catalyzing the stereoselectivehydrolysis of said mixture I to provide said mixture II, and effectingsaid conversion.
 18. The method of claim 17, wherein a mixture Icomprising the enantioners Ia(1) and Ib(1) is resolved: ##STR44## toform a mixture II comprising the compounds IIa(1) and IIb(1): ##STR45##19. The method of claim 18, wherein, in said mixture I comprising Ia(1)and Ib(1):R¹ is alkanoyloxy; R² is furyl or thienyl; and R³ is hydrogen,phenyl or substituted phenyl.
 20. The method of claim 19, wherein saidmixture I comprises (±)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one andsaid mixture II comprises (+)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-oneand (-)-cis-3-hydroxy-4-(2'-furanyl)azetidin-2-one.
 21. The method ofclaim 17, wherei said enzyme is immobilized on a support.
 22. The methodof claim 17, wherein, further, the non-enantiomeric compounds IIa andIIb of said mixture II obtained are separated by a separation step. 23.The method of claim 22, wherein said separation step is an extraction,distillation, crystallization, or column chromatography step.
 24. Themethod of claim 17, comprising the further step of preparing a taxanebearing a C-13 sidechain, wherein said sidechain is formed using atleast one of the non-enantiomeric compounds IIa or IIb of said mixtureII.
 25. A method for the resolution of a mixture I comprising theenantiomers Ia and Ib, where R¹ is in the cis position relative to R² inboth Ia and Ib, or where R¹ is in the trans position relative to R² inboth Ia and Ib: ##STR46## to form a mixture II comprising the compoundsIIa and IIb, where R^(1a) and R^(1b) are in the cis position relative toR² in both IIa and IIb, or where R^(1a) and R^(1b) are in the transposition relative to R² in both IIa and IIb, in each case correspondingto the position of R¹ in mixture I: ##STR47## where R¹ is hydroxyl;oneof R^(1a) or R^(1b) is hydroxyl and the other of R^(1a) or R^(1b) is R⁴--C(O)--O--, and R⁴ is alkyl or alkenyl; R² is furyl or thienyl; and R³is hydrogen or aryl;comprising the step of contacting said mixture I, inthe presence of a compound III:

    R.sup.4 --C(O)--L                                          (III)

where R⁴ is as defined above for R^(1a) or R^(1b) and L is a leavinggroup, with an enzyme which is selected from the group consisting of KIDlipase, lipase OF and Esterase 30,000, and which is capable ofcatalyzing the stereoselective esterification of said mixture I toprovide said mixture II, and effecting said conversion.
 26. The methodof claim 25, wherein a mixture I comprising the enantiomers Ia(1) andIb(1) is resolved: ##STR48## to form a mixture II comprising thecompounds IIa(1) and IIb(1): ##STR49##
 27. The method of claim 26,wherein, in said mixture II comprising IIa(1) and IIb(1):R^(1a) orR^(1b) is alkanoyloxy; R² is furyl or thienyl; and R³ is hydrogen,phenyl or substituted phenyl.
 28. The method of claim 27, wherein saidmixture I comprises (±)-cis-3-hydroxy-4-(2'-furanyl)azetidin-2-one, saidmixture II comprises (+)-cis-hydroxy-4-(2'-furanyl)azetidin-2-one and(-)-cis-3-acetoxy-4-(2'-furanyl)azetidin-2-one, and said compound of theformula III is isopropenyl acetate.
 29. The method of claim 25, whereinsaid enzyme is immobilized on a support.
 30. The method of claim 25,wherein, further, the non-enantiomeric compounds IIa and IIb of saidmixture II obtained are separated by a separation step.
 31. The methodof claim 30, wherein said separation step is an extraction,distillation, crystallization or column chromatography step.
 32. Themethod of claim 25, comprising the further step of preparing a taxanebearing a C-13 sidechain, wherein said sidechain is formed using atleast one of the non-enantiomeric compounds IIa or IIb of said mixtureII.